Methods for treating bacterial infections with phrenosin

ABSTRACT

A variety of substances are reported which alter host resistance to cocci bacterial infections. Nevertheless drugs which induce a high degree of resistance to these infections when administered prophylactically are not in wide use. Drugs which confer immunity to these infections for more than a week after administration are virtually unknown. Antimicrobials have been found which are effective in inducing resistance to infections due to cocci when administered prophylactically.

United States Patent inventors Elton S. Cook Cincinnati, Ohio; Norbert J. Berberich, Jr., Covington, Ky. App]. No. 709,937 Filed Mar. 4, 1968 Patented Nov. 23, 1971 Assignee Stanley Drug Products, Inc. Portland, Oreg.

METHODS F OR TREATING BACTERIAL INFECTIONS WITH PHRENOSIN Primary Examiner-Jerome D. Goldberg Attorney-Kinney and Schenk ABSTRACT: A variety of substances are reported which alter host resistance to cocci bacterial infections. Nevertheless drugs which induce a high degree of resistance to these infections when administered prophylactically are not in wide use. Drugs which confer immunity to these infections for more than a week after administration are virtually unknown. Antimicrobials have been found which are effective in inducing resistance to infections due to cocci when administered prophylactically.

METHODS FOR TREATING BACTERIAL INFECTIONS WITH PHRENOSIN BACKGROUND OF THE INVENTION This invention pertains to antimicrobials. In a particular aspect the invention relates to antimicrobials effective in protecting against cocci bacterial infections.

Bacteria such as cocci are a unique group of organisms embodying within themselves an array of yet unanswered puzzles in biology, both fundamental and experimental. They are ubiquitous in distribution and have attained extreme degrees of diversification in biological and biochemical characteristics. It is recognized that the significance of staphylococcal infections is not so much in severity, except in a few instances, as in the subtleties of the infection due to the unpredictable vagaries of these organisms.

Treatment of cocci bacterial diseases is complicated by the ability of the organisms to develop resistance. The magnitude of the problem is further amplified by the extreme difiiculty of total eradication, and the frequent reappearance of the same strain even after apparently successful elimination. The inability to eliminate the carrier state by any of the currently known methods and the prevalence of the new antibiotic-resistant hospital strains have added a new dimension to the frustrating situation.

Penicillin G (benzyl penicillin) is still the drug of choice for the treatment of infections caused by susceptible coccic strains. However numerous strains are known which elaborate enzymes such as penicillinase in response to the drugs and thus remain insensitive. This led to the development of semisynthetic penicillins which are not inactivated by penicillinase. However recently resistance of staphylococci to the newer penicillins has been reported. Hence there is a seemingly never ending demand for anticoccic and antibacillic factors.

A variety of substances are reported which alter host resistance to cocci infections. However, drugs which induce a high degree of resistance to coccic infections when administered prophylactically are not in wide use. Significantly, drugs which'confer immunity to coccic infections for more than a week after administration are virtually unknown. This invention provides an antimicrobial which not only expresses its anticoccic properties 1 hour after administration but which confers immunity to staphylococcal infections up to 4 weeks after administration. Even more remarkably an injection afier treatment gives complete immunity to reinfection up to at least 2 months.

SUMMARY OF THE INVENTION DETAILED DESCRIPTION OF THE INVENTION The processes of infection leading to disease are accepted to be a problem in the ecology of the parasite. It is being increasingly realized that the bacterial and host detemlinants are closely interrelated. Staphylococcal virulence derives from the combined action of several bacterial factors whose effectiveness is conditioned by the reactions of the host. Perhaps the most striking feature of host-parasite relationships in staphylococcal infections is the relatively atypical immunologic response. Human studies have given convincing evidence that most adult humans possess an array of antistaphylococcal antibodies. Nevertheless resistance to staphylococci seems to be governed to a considerable extent by other unrelated factors. For this reason attempts at prophylactic administration have not been completely successful.

In vitro experiments performed to study the effects on the best known characteristics of Staphylococcus aureus of the two sphingolipids concerned herein led to the conclusion that the sphingolipids contemplated do not alter the biochemical 5 characteristics of the organism. This is very significant indeed.

It means that in the true sense these compounds are not antibiotics. Antibiotics are somewhat poisonous, being more poisonous toward the organism than toward the host. The in vitro tests showed that these compounds do not kill the organism. However quite surprisingly, in the system of the host they create an environment in which the organism apparently cannot grow. Thus they unexpectedly render immunity to the host, but without the organism itself being present as it is in vaccines. The compositions of this invention thus constitute a 5 significant new class of antimicrobials. It is contemplated that they will be taken orally periodically like vitamins, or by intramuscular injection, or, say, 250 mg. per day dose, based on the chemical per se, oral administration desirably being one 400 mg. tablet or pill per day. An advantage of the invention is that prior to periods where contact with staphylococci infec' tions are more likely, such as prior to entering a hospital, injections or tablets will be used by the physician.

The high degree of resistance to staphylococcal infections obtained by the prophylactic administration of these sphingolipids will best be apparent from their biological effects in in vivo studies. Since both phrenosin and sphingomyelin are known, and are commercially available .compounds, their preparation need not be described herein. sphingomyelin and phrenosin are relatively insoluble in water, but they were emulsified by warming gently and shaking vigorously.

Throughout the course of these studies C3H, and Swiss albino mice, both male and female, were used. The animals were between and 30 weeks old and had an approximate average weight of to grams. These animals were mostly raised and maintained on the Rockland diet.

The assays were conducted using a penicillin-resistant strain, Staphylococcus aureus Original, first isolated from a case of acute tonsilitis and maintained in our laboratories for years. This strain is preserved in the lyophilized form and stored at 0 C. and stock cultures were raised on SA 1 l0 slants once in every 6 months. For testing, the inoculum was prepared from 24 hour cultures on SA 1 l0 slants at 37 C. The cells were washed and suspended in physiological saline (TC Tyrode Solution, Difco). In contrast to conventional procedures, an LD 80-90 instead of LD 50 was used in these investigations. This has been the practice in our laboratories in studies with staphylococci since lower dosages often fail to give adequate degrees of mortality. The LD 80-90 was determined by injecting groups of mice subcutaneously with different dilutions of the bacterial suspension and noting the mortality over a 5-day period.

Using six groups of C3H/HeJ female mice ranging in age from 10 to II weeks, a biological assay of sphingomyelin was conducted. The animals were inoculated subcutaneously for 3 consecutive days with various amounts of sphingomyelin. On the fourth day they were challenged with a suspension of the Original Strain organism giving a 60 percent transmission. The results recorded in table I were noted.

TABLE i [Sphlngornyellm prophylactic, subcutaneous] Percent mortality in hours When challenged with suspension of the Original Strain giving 55 percent transmission, the results in Table 2 were obtained.

TABLE 2 TABLE 6 [Sphingomyelin, prophylactic, subcutaneous] [Survivors rechallenged 41 days alter the first challenge] Percent mortality in hours Percent mortality in Dose pelr post-challenge 5 Number days post-challenge anlma, Group mg. 24 48 72 96 120 Group animals 1 2 3 4 5 80 Control 80 90 90 90 9o 0 Survivors from phrenosin... 5 0 0 0 0 0 These results demonstrate that sphingomyelin has I These rechallen g e experimcntsifiare performed at varyingconsiderable activity in ofl'setting the lethal efi'ects Pf 1O i terv ls afic [he first challenge up to a maximum of 59 days stilphylococcai i fi' T 9 i fg and it was consistently observed that irrespective of the treatwith phrenosm t e to owing 1s glven, Wlss mo ment prior to challenge, the survivors showed almost mlce bemg used' T ABLE 3 complete immunity at least up to the periods covered in our Percent mortality in days post- Dose per challeng animal, Number Group mg. of 1 2 3 4 5 animals Control 10 50 50 60 60 60 gphingomyelln 5 10 40 hrenosln 5 10 20 40 40 50 Control 10 50 S0 Sphingomyclin. 5 10 30 40 70 70 80 Phrenosin 5 10 10 30 40 40 40 These data not only illustrate the definite value of phrenosin experiments.

in protecting Swiss albino mice against experimental The compositions of this invention thus constitute a signifistaphylococcal infections, but show that phrenosin is even cant new class of antimicrobials. It is contemplated that they more effective than sphingomyelin. will be taken orally periodically like vitamins, say, weekly or It was decided to assay these compounds in different mouse 30 monthly, in 250 to 500 mg. tablets. Prior to periods where strains to ascertain whether discrepancies in results would apcontact with staphylococci or streptococci infections are more pear on the basis of host differences. Assays were conducted likely, such as prior to entering a hospital, injections of say on C3H females using sphingomyelin and phrenosin in a 3-day 150 to 500 mg. will be used by the physician. The sphingolipid prophylactic series. Table 4 shows the results of these runs. can be combined with an aqueous, vegetable oil,

TABLE 4 [Sphingomyelin and phrenosin in 03H mice, prophylactic] Percent mortality in days post- These studies show that host diiferences influence to some degree the in viva activity of the compounds assayed.

In view of the relatively frequent recurrence of staphylococmonoglyceride or diglyceride vehicle for injection, sodium cal infections, it is generally thought that immunity to this or- 50 hl ide b ing sed if ne e ary to render the solution ganism is short-lived or only partial. it was. the efo e, of isotonic. The suspension or solution will contain 0.1 to 5 perterest to see if the survivors from groups subjected to lethal ent, referably 0,1 to 1,5 percent of the sphingolipid by doses are immune after prophylactic administration to rechalweight. lenge. Survivors were from the groups which had been treated In the case of tablets, if desired, suitable colorants, adhewith sphingomyelin and phrenosin. Brain extract was also in- 55 sives, and lubricants will be incorporated along with a solid cluded in this work in order to obtain a comparative picture. h ti l dil t, for instance, starches, lactose,

The survivors after the first challenge in the respective sucrose and other pharmaceutical diluents. These tablets will groups were maintained under ideal conditions, and after apcontain 20 to 50 percent of the sphingolipid. Capsules can also propriate intervals they were rechallenged with the lethal dose be made. Thus a process is provided for the control of infecof Staphylococcus aureus. Fresh control groups were employed 60 tions in humans and other mammals due to cocci and bacilli each time. Mortality in each group was taken as an index of which involves administering to the mammal a prophylactirelative resistance or susceptibility as the case may be. Essencally effective amount of the sphingolipid. Various diluents, tially the same results were obtained in several of the runs. doses, and other variations and modifications will occur to The results of one such assay, reproduced in ta e 5, e those skilled in the art. Such ramifications are deemed to be representative of these runs. In this particular case, the 65 within the scope of this invention.

animals were raised on an experimental diet and were rechal- What is claimed is: lenged 59 days after the first challenge. 1. A method of treating bacterial Infections in mammals comprising administering to a mammal suffering from said bacterial infection an antibacterial efi'ective amount of I TABLE" 5 7O phrenosin. [smivms rechumted 59 days the first challenge] 2. The method of claim 1 wherein the bacterial infection is Percent mortality in due to a coccus. days 3. The method of claim 2 wherein the coccus is Staphylococ- Group animals 1 2 3 4 5 cus sureus. m m0 100 100 100 75 4- The method of claim 1 where n the phrenosin 1s m admix- Survivors from brain extrae tfn. 7 14.2 14.2 14. 2 14.2 14.2 ture with a solid pharmaceutical diluent.

urvlvors from sphingomye n. 5 0 0 0 0 0 sumvom from phmmmnfl 5 0 o 0 0 0 5. The method of claim 1 wherein the phrenosin IS in the form of an aqueous suspension. 

2. The method of claim 1 wherein the bacterial infection is due to a coccus.
 3. The method of claim 2 wherein the coccus is Staphylococcus sureus.
 4. The method of claim 1 wherein the phrenosin is in admixture with a solid pharmaceutical diluent.
 5. The method of claim 1 wherein the phrenosin is in the form of an aqueous suspension. 